The overall objective of the proposed Mentored Quantitative Research Career Development Award (K25) is to provide a period of mentored didactic and research training that will allow Dr. Krishanu Ray with his physics background to make a career transition into an independent biomedical researcher. The immediate goal is for Dr. Ray to expand his current research program in applying advanced fluorescence spectroscopy techniques to interrogate virus-host interactions in early stages of HIV infection. Specifically, Dr. Ray will employ novel fluorescence based approaches not previously described for viral entry process. The four-year training plan involves a structured curriculum of coursework and seminars focusing on HIV related research. This program will be heavily weighted toward gaining expertise regarding the HIV biology, particularly using optical imaging and spectroscopy based approaches to HIV research under the mentorship of two exceptional scientists, Dr. Joseph R. Lakowicz and Dr. Anthony L. DeVico at the University of Maryland School of Medicine (UMSM). Because of Dr. Ray's location in the Center for Fluorescence Spectroscopy (CFS) and adjacent to the Institute of Human Virology (IHV) at the UMSM, it is possible to study the biophysical properties of HIV proteins and neutralizing antibody binding against HIV-1 envelope. The long-term goal is for Dr. Ray to acquire advanced biomedical research skills and knowledge that complement his skills as a spectroscopist. The ultimate goal is for him to develop into an independent, interdisciplinary biomedical researcher. This K25 award will enable Dr. Ray to take advantage of the outstanding resources available at CFS and IHV and expand his expertise and experience in this unique multidisciplinary setting. Dr. Ray will develop methods applying fluorescence correlation spectroscopy (FCS) and single molecule detection (SMD) to directly probe the nature of HIV-1 envelope interactions with cell surface receptors and/or anti-envelope antibodies at the molecular level. It is hypothesized that a detailed understanding of the conformational dynamics that impact the HIV envelope before and during receptor engagement will explain the observed susceptibility to various inhibitors. Accordingly, Dr. Ray will focus on the following aims: 1) To develop methods for examining HIV-1 gp120-CD4 interactions at the single molecule level. 2) To characterize HIV-1 Env trimer conformational dynamics using single molecule spectroscopy. 3) To compare the binding of human monoclonal antibodies with different neutralizing potencies to HIV-1 envelope trimers by FCS and SMD. Successful completion of the Aims will provide new quantitative approaches at the individual molecular level towards understanding early steps in viral entry. This program will provide an excellent milieu in which the applicant - under close mentorship - can become deeply involved in applying advanced fluorescence spectroscopic and imaging approaches to HIV research. Mentorship will be geared more toward biological issues to provide a well-rounded portfolio as the candidate progresses toward independence in biomedical spectroscopic and imaging research. Human-immunodeficiency virus type 1 (HIV-1) has been the subject of intensive research. The majority of antiretroviral drugs acts at a post-infection step and cannot cure HIV-1 infection. Consequently, there is a continued emphasis on developing either drugs or vaccines that block pre-entry events by targeting the HIV-1 surface trimers which enable binding to cell surface coreceptors. In this application, the nature of HIV-1 envelope interactions with cell surface receptors and/or anti-envelope antibodies will be determined at the single molecule level. This novel application of single molecule fluorescence based approaches is expected to provide unique information relevant to vaccine and drug development.